The long term objective of this proposal is to understand how entry into mitosis is regulated during a normal cell cycle (mitotic control) and how entry into mitosis is prevented when unreplicated or damaged DNA is detected (G2 checkpoint control). In eukaryotes, entry into mitosis is regulated by the synergistic and opposing activities of several distinct protein kinases and protein phosphatases. This cascade converges on Cdc2, a serine/threonine protein kinase required for the entry of cells into mitosis. Two neighboring amino acids within the amino terminus of Cdc2 (threonine 14 and tyrosine 15) have been shown to be critical elements in regulating the kinase activity of Cdc2. Phosphorylation of these residues maintains Cdc2 in an inactive state until the appropriate time in the cell cycle. In humans, the Weel and Myt1 protein kinases regulate the phosphorylation of Cdc2 on Tyr 15 and Thr 14 whereas the Cdc25C protein phosphatase dephosphorylates Cdc2 on both Thr 14 and Tyr15. Both Weel and Cdc25C interact with 14-3-3 proteins and in the case of Cdc25C, 14-3-3p binding negatively regulates the functional interactions between Cdc25C and Cdc2. Studies aimed at elucidating how the Weel and Myt1 kinases are regulated throughout the cell cycle are proposed. In addition, the Weel and Myt1 kinases will be functionally distinguished in human cells. The contribution made by 14-3-3 proteins to G2 checkpoint control will be examined in both mammalian systems and in the fission yeast, Schizosaccharomyces pombe. Finally studies will be performed to elucidate the mechanistic basis of G2 cell cycle arrest following DNA damage because many cancers are neither curable using existing strategies nor readily detachable at the earl stages there is a need to identify new targets that can be used both as diagnostic probes and as therapeutic targets. The studies outline in this proposal investigate basic mechanisms of cell cycle control and G2 checkpoint control. Proteins involved in these pathways may on day be used as diagnostic markers or as targets for designing anti-proliferative drugs.